The calcium-sensing receptor (CaR) is a G-protein coupled receptor that controls mammalian extracellular calcium (Ca2+o) homeostasis. CaR downstream signalling involves intracellular calcium (Ca2+i) mobilisation which can be negatively modulated by protein kinase C (PKC)-mediated phosphorylation of CaR residue Thr-888 (CaRT888). The nature of this regulation was investigated here using siRNA-based knockdown of individual PKC isotypes. Knocking down PKCα expression increased CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly lowering the EC50 for Ca2+o relative to control siRNA-transfected cells. In accordance, PKCα knockdown also decreased CaRT888 phosphorylation which also permitted the triggering of Ca2+i mobilisation in CaR-HEK cells at sub-threshold Ca2+o concentrations. Interestingly, PKCε knockdown attenuated CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly increasing the EC50 for Ca2+o. However, this knockdown was also also found to inhibit CaRT888 phosphorylation and this is the first time that CaRT888 phosphorylation has been shown to be dissociate from Ca2+i mobilisation. The results show the complexity of the interactions that potentially underlie the CaR’s pleiotropic signalling and provides novel targets for examining signal bias. Classically an increase in cAMP is known to trigger PTH seceretion. The observation in this study shows that raising intracellular cAMP levels with forskolin also decreased CaRT888 phosphorylation permitting increased Ca2+i mobilisation. This suggests that cAMP may stimulate the phosphatase (most likely protein phosphatase 2A (PP2A)). Nevertheless, knocking down Gα12, which has been shown to activate PP2A, resulted in increased CaRT888 phosphorylation and lower Ca2+i mobilisation (increased EC50 for Ca2+o). This suggests the possibility of CaR as a cAMP sensor that can detect an increase in intracellular cAMP in order to stop PTH serection. Three novel CaR effectors, P70 ribosamal protein S6 kinase, insulin-like growth factor receptor-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, were identified in CaR-HEK cells. It was shown that a) high Ca2+o stimulated the activation of these effectors and b) each effector was inhibited by knockdown of PKCα and Gα12, which further confirmed the association of these signals with CaR. These data show that CaR also plays an important role outside Ca2+o homeostasis, such as growth and inflammation. Finally, five CaR mutations associated with autosomal dominant hypocalcaemia (ADH) were found to increase Ca2+o-induced Ca2+i mobilisation, as well as ERK and p38MAPK activation, when transfected stably in HEK-293 cells. Cotreatment with the calcilytic NPSP795 inhibited ERK and p38MAPK phosphorylation in all 5 gain-of-function mutants and in the wild type CaR cells, with IC50s for the compound in the nanomolar range. These data highlight the potential utility of CaR negative allosteric modulators in the treatment of gain-of-function CaR mutations. Together these data enhance our understanding of CaRT888 phosphorylation and CaR signalling.

It is now accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate and calcium homeostasis through its membrane receptor termed the 1,25D3-MARRS (membrane associated rapid response steroid binding) protein. I determined the various PKC isotypes involved in the rapid regulation of phosphate uptake and calcium extrusion in chick intestinal cells. 1,25(OH)2D3-mediated phosphate uptake was stimulated within 1 min after addition of the hormone. Western blot analyses on isolated intestinal cells treated with steroid hormone resulted in dose-dependent increases in PKC alpha and PKC beta in postnuclear centrifugation fractions, but not in the low speed centrifugation fractions. The highest immunoreactivity of PKC alpha was found after treatment of the cells with 300 pM 1,25(OH)2D3 and declined at 650 pM hormone, relative to corresponding controls, while the highest immunoreactivity of PKC beta was found in cells treated with either 300 pM or 650 pM 1,25(OH)2D3. Therefore, PKC alpha and PKC beta redistribution are likely to relate to the dose-response curve for both phosphate uptake and calcium efflux, respectively. Using transfection of primary cultures of intestinal cells with siRNA for these two isotypes, I found decreased 32P uptake in cells transfected with siRNA to either PKC alpha or PKC beta in both controls (relative to untransfected controls), and hormone-treated cells. Further study of the effect of chemical blockers for PKC alpha or PKC beta on phosphate uptake was conducted in suspensions of isolated intestinal cells. The results from these experiments also confirmed the findings from the siRNA experiments and demonstrated decreased 32P uptake in cells treated with 1,25(OH)2D3 plus blockers in comparison with cells treated with 1,25(OH)2D3 alone. The effects of PKC alpha and PKC beta in steroid-mediated calcium extrusion were further investigated using siRNA for PKC alpha or PKC beta. We found the siRNA to PKC beta alone caused decreased calcium extrusion. We also found that the inhibitors of PKC beta, but not PKC alpha caused significantly enhanced calcium uptake by decreasing calcium efflux from the cells. This result suggested that PKC beta might be involved in the rapid response of 1,25(OH)2D3-stimulated calcium extrusion. I used confocal microscopy to study the redistribution of PKC alpha and PKC beta in cells exposed to steroid hormone for 30 sec. PKC alpha was found to increase significantly in the apical membrane after a 30 sec exposure of cells to 300- or 650 pM 1,25(OH)2D3. By comparison, anti-PKC beta immunofluorescence was found to increase significantly in the basal region of cells, relative to controls, following exposure of cells to 300 pM seco-steroid. These combined results, lead me to conclude the involvement of both PKC alpha and PKC beta in the signal transduction mechanism of 1,25(OH)2D3-mediated phosphate uptake while PKC beta is involved in the mechanism of 1,25(OH)2D3-mediated calcium efflux in chick intestinal epithelial cells.